Enzymatic process for the preparation of (1s,2r)-2-(difluoromethyl)-1-(propoxycarbonyl)cyclopropanecarboxylic acid

ABSTRACT

Disclosed are methods of synthesizing enantioenriched difluoroalkylcyclopropyl amino esters and their salts, such as the dicyclohexylamine salt of (1S,2R)-2-(difluoromethyl)-1-(propoxycarbonyl)cyclopropane carboxylic acid. These compounds are useful intermediates in the synthesis of viral protease inhibitors.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/010,557, filed Jan. 29, 2016, now U.S. Pat. No. 10,316,338, whichclaims the benefit of priority to U.S. Provisional Patent ApplicationSer. No. 62/109,943, filed Jan. 30, 2015.

BACKGROUND

Complex biologically active molecules are challenging, expensive, andtime-consuming to synthesize. Synthesizing chiral, non-racemic compoundswith good enantio- and diastereoselectivity is even more challenging.Doing so generally involves isolating or synthesizing an enantioenrichedintermediate whose stereochemistry can be preserved in the requiredsubsequent synthetic transformations.

An example of a useful intermediate in the synthesis of a biologicallyactive molecule is(1R,2R)-1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropanecarboxylicacid (1, Boc-DFAA). In the past, this intermediate was synthesized from(1R,2S)-2 using corrosive fluorination chemistry, which is not suitablefor large scale production. WO 2009/064975.

There exists a need for new synthetic methods to constructenantioenriched difluoroalkylcyclopropyl amino esters and theirprecursors.

SUMMARY OF THE INVENTION

In certain embodiments, the invention relates to a method according toreaction Scheme A:

wherein R is alkyl.

In some embodiments, the invention relates to any of the methodsdescribed herein, wherein the first solvent is preferably an aqueoussolution of sodium citrate or calcium acetate at a concentration of fromabout 0.05 M to about 0.15 M.

In certain other embodiments, the invention relates to any of themethods described herein, wherein the first enzyme is preferably lipasefrom Thermomyces lanuginosus (AH-45) or (Rhizo-)Mucor miehei (RML).

In certain embodiments, the invention relates to a method according toreaction Scheme B:

wherein R is alkyl.

In some embodiments, the invention relates to any of the methodsdescribed herein, wherein the second solvent is preferably an aqueoussolution of sodium phosphate at a concentration of from about 0.05 M toabout 0.15 M.

In certain other embodiments, the invention relates to any of themethods described herein, wherein the second enzyme is preferably yvaKesterase or BsteE esterase.

In certain embodiments, the invention relates to a method according toreaction Scheme C:

wherein R is alkyl.

In some embodiments, the invention relates to any of the methodsdescribed herein, wherein the third solvent preferably comprises anaqueous solution of monopotassium phosphate at a concentration of fromabout 0.25 M to about 0.75 M.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the third solvent further comprisestetrahydrofuran (THF), methyl tert-butyl ether, ethyl acetate, dioxane,DMF, acetonitrile, or DMSO, preferably methyl tert-butyl ether.

In certain other embodiments, the invention relates to any of themethods described herein, wherein the third enzyme is preferably yvaKesterase or BsteE esterase.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein R is preferably ethyl, propyl, or butyl.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

In certain embodiments, the invention relates to a method ofsynthesizing enantioenriched compounds, such as(1S,2R)-2-(difluoromethyl)-1-(propoxycarbonyl)cyclopropane carboxylicacid (6), by selective enzymatic hydrolysis. The inventive methods aremore efficient than known methods because (i) they preferably do notinvolve synthesizing a racemate and separating enantiomers, and (ii)enantioenriched starting materials are not required.

In certain embodiments, the invention relates to a method ofsynthesizing a Drug Substance via(1R,2R)-1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropanecarboxylic acid (1) as shown in Scheme 2.

In certain embodiments, the methods of the invention are based on theenzymatic reactive resolution of (±)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (7) followed by enzymaticdesymmetrization of the resulting unreacted (R)-diester ((R)-7) toafford (1S,2R)-2-(difluoromethyl)-1-(propoxycarbonyl) cyclopropanecarboxylic acid (6), which is isolated as its dicyclohexylamine salt(8), as described in Scheme 3 and Scheme 4.

In one embodiment, the (1S,2R)-mono-acid DCHA salt (8) is converted to(1R,2R)-1-((tert-butoxycarbonyl) amino)-2-(difluoromethyl)cyclopropanecarboxylic acid (1, Boc-DFAA) via Curtius Rearrangementfollowed by hydrolysis, as in Scheme 5.

In certain preferred embodiments, the methods do not involve corrosivefluorination reagents or laborious and expensive Simulated Moving Bed(SMB) chromatography for the separation of the desired chiral isomer.

In certain preferred embodiments, the methods improve processability ofthe isolated intermediates.

Preferably, the overall yield of(1R,2R)-1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropanecarboxylic acid (1) is significantly improved as compared to knownprocesses.

II. Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The number of carbon atoms in a hydrocarbyl substituent can be indicatedby the prefix “C_(x)-C_(y),” where x is the minimum and y is the maximumnumber of carbon atoms in the substituent.

The term “alkyl” as used herein, refers to a saturated, straight- orbranched-chain hydrocarbon radical typically containing from 1 to 20carbon atoms. For example, “C₁-C₆ alkyl” or “C₁-C₈ alkyl” contains fromone to six, or from one to eight, carbon atoms, respectively. Examplesof alkyl substituents include, but are not limited to, methyl, ethyl,propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl,octyl substituents and the like.

The term “cycloalkyl” denotes a monovalent group derived from amonocyclic or polycyclic saturated carbocyclic ring compound. Examplesof cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyland the like.

The term “amino-protecting group,” as used herein, refers to a labilechemical moiety that can protect an amino group against undesiredreactions during synthetic procedures. After said syntheticprocedure(s), the amino-protecting group as described herein may beselectively removed. Suitable amino-protecting groups are describedgenerally in T. H. Greene and P. G. M. Wuts, Protective Groups inOrganic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).Examples of amino-protecting groups include, but are not limited to,t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and thelike.

The term “protected amino,” as used herein, refers to an amino groupprotected with an amino-protecting group as defined above.

As used herein, the term “salt” includes “pharmaceutically acceptablesalts,” which are, within the scope of sound medical judgment, suitablefor use in contact with the tissues of humans and other vertebrates,preferably mammals, without undue toxicity, irritation, allergicresponse and the like, and are commensurate with a reasonablebenefit/risk ratio. Pharmaceutically acceptable salts are well known inthe art. For example, S. M. Berge, et al. describe pharmaceuticallyacceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19(1977). Such salts can be prepared in situ during isolation andpurification of reaction products as described herein, or separately,such as by reacting a free base function with a suitable acid, such asan organic acid. Examples of pharmaceutically acceptable salts include,but are not limited to, hydrochloride, hydrobromide, phosphate, sulfate,perchlorate, acetate, maleate, tartrate, citrate, succinate, ormalonate. Other pharmaceutically acceptable salts include, but are notlimited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,sulfate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, or magnesium salts, and thelike. Further pharmaceutically acceptable salts include, whenappropriate, ammonium, quaternary ammonium, and amine cations associatedwith counterions such as halide, hydroxide, carboxylate, sulfate,phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate andaryl sulfonate.

As used herein, the term “enantioenriched” means a mixture ofenantiomers in which one of the two enantiomers is present in a largeramount (e.g., having an enantiomeric excess (ee) greater than about 90%,greater than about 95%, preferably greater than about 98%, mostpreferably greater than 99%). This term also encompasses anenantiomerically pure compound.

Various aspects of the invention are described in further detail herein.

III. Exemplary Methods and Uses

The compounds and processes of the present invention will be betterunderstood in connection with the following illustrative methods bywhich the compounds of the invention may be prepared. It will beunderstood that any reaction described herein, in any of its variations,can be combined in sequence with one or more of the other reactionsdescribed herein, in any of their variations, substantially in analogywith the sequence shown in the Schemes.

In certain embodiments, the invention relates to a method comprising areactive resolution according to reaction Scheme A:

wherein R is alkyl.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the first solvent is an aqueous buffer, suchas an aqueous solution of sodium citrate or calcium acetate, e.g., at aconcentration of from about 0.05 M to about 0.15 M, for example, about0.05 M, about 0.06 M, about 0.07 M, about 0.08 M, about 0.09 M, about0.10 M, about 0.11 M, about 0.12 M, about 0.13 M, about 0.14 M, or about0.15 M, preferably about 0.1 M.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the first solvent is an aqueous solution ofsodium citrate, e.g., at a concentration of from about 0.05 M to about0.15 M, for example, about 0.05 M, about 0.06 M, about 0.07 M, about0.08 M, about 0.09 M, about 0.10 M, about 0.11 M, about 0.12 M, about0.13 M, about 0.14 M, or about 0.15 M, preferably about 0.1 M.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the first pH is from about 5 to about 8.5, forexample, about 5, about 5.25, about 5.5, about 5.75, about 6, about6.25, about 6.5, about 6.75, about 7.0, about 7.25, about 7.5, about7.75, about 8.0, about 8.25, or about 8.5, preferably about 5.75. Incertain embodiments, the pH is from about 5 to about 6.5, for example,about 5, about 5.25, about 5.5, about 5.75, about 6, about 6.25, orabout 6.5, preferably about 5.75.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the first enzyme is a hydrolase, such as alipase, preferably from Thermomyces lanuginosus (AH-45) or (Rhizo)-Mucormiehiri (RML). In certain embodiments, the first enzyme is a lipase fromThermomyces lanuginosus (AH-45).

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the loading of the first enzyme is from about50 wt % to about 150 wt % as compared to the starting material, forexample, about 50 wt %, about 60 wt %, about 70 wt %, about 80 wt %,about 90 wt %, about 100 wt %, about 110 wt %, about 120 wt %, about 130wt %, about 140 wt %, or about 150 wt %, preferably about 100 wt % ascompared to starting material.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the first temperature is from about 10° C. toabout 40° C., for example, about 15° C., about 20° C., about 25° C.,about 30° C., about 35° C., or about 40° C., preferably about 20° C.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the first period of time is from about 36 h toabout 100 h, for example, about 36 h, about 40 h, about 44 h, about 48h, about 52 h, about 56 h, about 60 h, about 64 h, about 68 h, about 72h, about 76 h, about 80 h, about 84 h, about 88 h, about 92 h, or about98 h, preferably about 72 h.

In certain embodiments, the invention relates to any of the methodsdescribed herein, further comprising crystallizing the diester reactionproduct of reaction Scheme A to obtain the diester compound in acrystalline form.

In certain embodiments, the invention relates to any of the methodsdescribed herein, further comprising separating the hydrolyzed reactionproduct of reaction Scheme A from the reaction mixture.

In certain embodiments, the invention relates to any of the methodsdescribed herein, further comprising isolating the diester reactionproduct of reaction Scheme A from the reaction mixture, therebyobtaining substantially pure diester reaction product of Scheme A.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the enantiomeric excess of the diesterreaction product is greater than about 90%, for example, about 91% ee,about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee,about 97% ee, about 98% ee, about 99% ee, preferably greater than about94% ee.

In certain embodiments, the invention relates to a method comprisingdesymmetrizing a diester according to reaction Scheme B:

wherein R is alkyl.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the second solvent is an aqueous buffer, suchas an aqueous solution of sodium phosphate, e.g., at a concentration offrom about 0.05 M to about 0.15 M, for example, about 0.05 M, about 0.06M, about 0.07 M, about 0.08 M, about 0.09 M, about 0.10 M, about 0.11 M,about 0.12 M, about 0.13 M, about 0.14 M, or about 0.15 M, preferablyabout 0.1 M.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the second pH is from about 7.75 to about9.25, for example, about 8, about 8.25, about 8.5, about 8.75, or about9, preferably about 8.5.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the second enzyme is yvaK esterase, preferablyyvaK esterase from Bacillus subtilis.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the second enzyme is BsteE esterase,preferably BsteE esterase from Bacillus stearothermophilus.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the second enzyme is provided in a whole cell,such as a freeze-dried whole cell.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the loading of the freeze-dried whole cells isfrom about 40 wt % to about 150 wt % as compared to the startingmaterial, for example, about 40 wt %, about 45 wt %, about 50 wt %,about 55 wt %, about 60 wt %, about 65 wt %, about 70 wt %, about 75 wt%, about 80 wt %, about 85 wt %, about 90 wt %, about 95 wt %, about 100wt %, about 105 wt %, about 110 wt %, about 115 wt %, about 120 wt %,about 125 wt %, about 130 wt %, about 135 wt %, about 140 wt %, about145 wt %, or about 150 wt %, preferably about 85 wt % as compared tostarting material.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the second temperature is from about 10° C. toabout 40° C., for example, about 15° C., about 20° C., about 25° C.,about 30° C., about 35° C., or about 40° C., preferably about 20° C.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the second period of time is from about 10 hto about 30 h, for example, about 10 h, about 11 h, about 12 h, about 13h, about 14 h, about 15 h, about 16 h, about 17 h, about 18 h, about 19h, about 20 h, about 21 h, about 22 h, about 23 h, about 24 h, about 25h, about 26 h, about 27 h, about 28 h, about 29 h, or about 30 h,preferably about 21 h.

In certain embodiments, the invention relates to any of the methodsdescribed herein, further comprising isolating the reaction product ofreaction Scheme B from the reaction mixture, thereby obtainingsubstantially pure reaction product of reaction Scheme B.

In certain embodiments, the invention relates to any of the methodsdescribed herein, further comprising crystallizing the reaction productof reaction Scheme B to obtain the compound in a crystalline form.

In certain embodiments, the invention relates to any of the methodsdescribed herein, further comprising contacting the reaction product ofreaction Scheme B with a base to obtain a salt of the compound,optionally in a crystalline form. In some embodiments, the base is anamine, for example, a secondary amine, preferably dicyclohexylamine ordibenzylamine.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the enantiomeric excess of the reactionproduct of reaction Scheme B is greater than about 90%, for example,about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee,about 96% ee, about 97% ee, about 98% ee, about 99% ee, preferablygreater than about 96% ee.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the enantiomeric excess of the salt of thereaction product of reaction Scheme B is greater than about 90%, forexample, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee,preferably greater than about 97% ee.

In certain embodiments, the invention relates to a method comprising adesymmetrization according to reaction Scheme C:

wherein R is alkyl.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the third solvent comprises an aqueous buffer,such as an aqueous solution of monopotassium phosphate, e.g., at aconcentration of from about 0.25 M to about 0.75 M, for example, about0.25 M, about 0.3 M, about 0.35 M, about 0.4 M, about 0.45 M, about 0.5M, about 0.55 M, about 0.6 M, about 0.65 M, about 0.7 M, or about 0.75M, preferably about 0.5 M.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the third solvent further comprisestetrahydrofuran (THF), methyl tert-butyl ether, ethyl acetate, dioxane,DMF, acetonitrile, or DMSO, preferably methyl tert-butyl ether.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the third solvent comprises a mixture of anaqueous buffer and an organic solvent.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the third pH is from about 6.25 to about 7.75,for example, about 6.5, about 6.75, about 7.0, about 7.25, or about 7.5,preferably about 7.0.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the third enzyme is yvaK esterase, such asyvaK esterase from Bacillus subtilis.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the third enzyme is BsteE esterase, preferablyBsteE esterase from Bacillus stearothermophilus.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the loading of the third enzyme is from about40 wt % to about 150 wt % as compared to the starting material, forexample, about 40 wt %, about 45 wt %, about 50 wt %, about 55 wt %,about 60 wt %, about 65 wt %, about 70 wt %, about 75 wt %, about 80 wt%, about 85 wt %, about 90 wt %, about 95 wt %, about 100 wt %, about105 wt %, about 110 wt %, about 115 wt %, about 120 wt %, about 125 wt%, about 130 wt %, about 135 wt %, about 140 wt %, about 145 wt %, orabout 150 wt %, preferably about 70 wt % as compared to startingmaterial.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the third temperature is from about 10° C. toabout 40° C., for example, about 15° C., about 20° C., about 25° C.,about 30° C., about 35° C., or about 40° C., preferably about 20° C.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the second period of time is from about 50 hto about 150 h, for example, about 50 h, about 60 h, about 70 h, about80 h, about 90 h, about 100 h, about 110 h, about 120 h, about 130 h,about 140 h, or about 150 h, preferably about 100 h.

In certain embodiments, the invention relates to any of the methodsdescribed herein, further comprising isolating the reaction product ofreaction Scheme C from the reaction mixture, thereby obtainingsubstantially pure reaction product of reaction Scheme C.

In certain embodiments, the invention relates to any of the methodsdescribed herein, further comprising crystallizing the reaction productof reaction Scheme C to obtain the compound in a crystalline form.

In certain embodiments, the invention relates to any of the methodsdescribed herein, further comprising contacting the reaction product ofreaction Scheme C with a base to obtain a salt of the compound,optionally in a crystalline form. In some embodiments, the base is anamine, for example, a secondary amine, preferably dicyclohexylamine ordibenzylamine.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the enantiomeric excess of the reactionproduct of reaction Scheme C is greater than about 90%, for example,about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee,about 96% ee, about 97% ee, about 98% ee, about 99% ee, preferablygreater than about 98% ee.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein the enantiomeric excess of the salt of thereaction product of reaction Scheme C is greater than about 90%, forexample, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee,preferably greater than about 97% ee.

In certain embodiments, the invention relates to any of the methodsdescribed herein, wherein R is ethyl. In other embodiments, R is propyl,such as n-propyl. In yet other embodiments, R is butyl, such as n-butyl.

In certain embodiments, the invention relates to methods comprising twoor more of the steps described herein.

In certain embodiments, the invention relates to the use of any of thecompounds described herein in the manufacture of a medicament.

Definitions of variables in the structures in the schemes herein arecommensurate with those of corresponding positions in the formulaedelineated herein.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids.Optical isomers may be prepared from their respective optically activeprecursors by the procedures described above, or by resolving theracemic mixtures. The resolution can be carried out in the presence of aresolving agent, by chromatography or by repeated crystallization or bysome combination of these techniques which are known to those skilled inthe art. Further details regarding resolutions can be found in Jacques,et al., Enantiomers. Racemates, and Resolutions (John Wiley & Sons,1981).

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. In addition, the solvents, temperatures, reaction durations,etc. delineated herein are for purposes of illustration only and one ofordinary skill in the art will recognize that variation of the reactionconditions can produce the desired products of the present invention.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the compoundsdescribed herein are known in the art and include, for example, thosesuch as described in R. Larock, Comprehensive Organic Transformations,VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groupsin Organic Synthesis, 2d. Ed., John Wiley and Sons (1991): L. Fieser andM. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995), and subsequenteditions thereof.

EXEMPLIFICATION

The present invention is further illustrated by the following Examplewhich should not be construed as limiting in any way. The Examples anddiscoveries described herein are representative. As such, the studiesand results described in the Examples section herein may be used as aguideline.

Example 1: Knoevenagel Condensation

Magnesium chloride (5.06 g, 0.0531 mol, 0.05 equiv.) followed by1-propanol (600 mL, 3.0 mL/g of dipropyl malonate) were charged to aclean/dry 1.0 L glass reactor under nitrogen, which was set up with amechanical stirrer and thermocouple at ambient temperature. Agitationwas started and difluoroacetaldehyde ethyl hemiacetal (163.77 g, 1.1689mol, 1.10 equiv., based on 90% potency) was charged and the containerwas rinsed with 1-propanol (100 mL, 0.5 mL/g of dipropyl malonate). Tothis mixture, dipropyl malonate (DPM) (200.0 g, 1.0626 mol, 1.00 equiv.)was charged and the flask was rinsed with 1-propanol (100 mL, 0.5 mL/gof dipropyl malonate). The reaction mixture was heated at 60° C. for 46h and the reaction was deemed completion, as determined by GC analysisof in-process sample [Residual dipropyl malonate: 3.65%, and combinedmixture of dipropyl 2-(2,2-difluoro-1-propoxyethyl)malonate (11a) anddipropyl 2-(2,2-difluoroethylidene)malonate (11b): 79.54%]. The mixturewas cooled and distilled on a rotary evaporator at NMT 60° C. undervacuum to remove 1-propanol and other volatiles (Bath temp: 60° C.). Tothe resulting oily residue, MTBE (400 mL) was charged and thedistillation was continued under vacuum (Bath temp: 50° C.). MTBE chasedistillation was performed two more times (2×400 mL) for a total threetimes (400 mL MTBE each time) on rotary evaporator (Bath temp: 50° C.).After chase distillations were complete, the resulting oil (383.7 g,hazy solution and contains white solids) was suspended in MTBE (600 mL),mixed for 5 min, filtered and the solid was rinsed with MTBE (200 mL).The combined filtrate was distilled on a rotary evaporator (Bath temp:NMT 50° C.) to afford 351.6 g of dipropyl2-(2,2-difluoro-1-propoxyethyl)malonate, as a major product, which alsocontain dipropyl 2-(2,2-difluoroethylidene)malonate as minor product.Purity of the combined mixture by GC: 88.31% and crude product (mixtureof dipropyl 2-(2,2-difluoro-1-propoxyethyl)malonate (11a), and dipropyl2-(2,2-difluoroethylidene)malonate) (11b) used “as is” incyclopropanation step.

Example 2: Cylopropanation to form (±)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (7)

Potassium tert-butoxide (150.6 g, 1.2751 mol, 1.2 equiv.) followed by1000 mL of DMF were charged under nitrogen into a clean/dry 4 L glassreactor, which was set up with a mechanical stirrer, thermocouple andnitrogen inlet, and the agitation was started. Trimethylsulfoxoniumiodide (280.6 g, 1.2751 mol, 1.2 equiv.) was charged and the funnel wasrinsed with 400 mL of DMF under nitrogen. The mixture was agitated for 1h at 20° C. temperature under nitrogen. The crude (dipropyl2-(2,2-difluoro-1-propoxyethyl)malonate (11a) and dipropyl2-(2,2-difluoroethylidene)malonate) (11b) was diluted with DMF (150 mL),was charged under nitrogen. The container was rinsed DMF (50 mL) and themixture was heated at 55° C. for 3 h under nitrogen. Based on GCanalysis in-process samples, the reaction was deemed complete with 1.19%of residual dipropyl 2-(2,2-difluoroethylidene)malonate (11b) at 17.28min and 94.33% of (±)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (7) at 15.29 min. Themixture was cooled to 0-5° C. and the reaction was quenched with water(2.0 L) and MTBE was charged (2.2 L). The temperature of the contentswas raised to 20° C. and mixed for 15 min. The aqueous and organicphases were separated and the aqueous phase was back extracted with MTBEtwo times (1.8 L and 1.0 L). The combined organic phase was washed with20% sodium chloride soln three times (3×1.25 L), dried with anhydrousmagnesium sulfate (50 g), and filtered. The filtrate was concentrated ona rotary evaporator (Bath temp: 55° C.) to afford 278.92 g of(±)-dipropyl 2-(difluoromethyl)cyclopropane-1,1-carboxylate (7), as ayellow/pale red oil with 94.89% (pa) purity by GC.

A portion of the crude (±)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (7, 61.3 g) was purifiedby flash chromatography (TLC: Hexanes: Rf: 0.2 (Ethyl acetate/10:90,Permanganate strain) on CombiFlash RF system using pre-packed RediSepRf, Silica column (330 g) and mixture of hexanes and ethyl acetatesolvent (90:10) at flow rate of 220 mL/min. The desired fractions werecombined (A 16-30, B1-30, C1-30, and D 1-11), and the combined fractionswere concentrated on rotary evaporator (Bath temp: 60° C.) and finallychase distilled with hexanes (3×600 mL) on rotary evaporator (Bath temp:60° C.) to afford 47.9 g (Colorless/clear oil) of (±)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (7) with 97.76% (pa)purity by GC, which used for initial enzyme screening reactions.

Example 3: One-Step Enzymatic Reactive Resolution and Desymmetrizationto Form(1S,2R)-2-(difluoromethyl)-1-(ethoxycarbonyl)cyclopropanecarboxylic acid(14, Free Acid)

Example 3a: Screen for Enzymes Capable of Reactively Resolving andDesymmetrizing 13

A panel of commerically-available lipases and esterases was screenedusing chiral HPLC to access their utility in the resolution anddesymmetrization of (±)-diethyl diester 13. Fourteen enzymes wereevaluated using the following procedure: Enzyme solutions were createdby dissolving 5 mg of lyophilized enzyme or 5 μL of liquid enzymesolution into 1 mL of 0.5 M sodium phosphate buffer with 0.2 M sodiumchloride at pH 8.0. Diester substrate solution was prepared by combining18 mg of (±)-diethyl diester 13 per mL of 0.5 M sodium phosphate bufferwith 0.2 M sodium chloride at pH 8.0. Reactions were initiated bycombining 800 μL of the diester substrate solution with 200 μL of theenzyme solution. The reactions were incubated at 30° C. and 225 rpm forapproximately 18 hours. Following incubation, reactions were preparedfor chiral HPLC by adding 120 mg sodium chloride and 20 μL of 5 Nhydrochloric acid. Two mL of methylene chloride were added to eachreaction, and each was vortexed, and centrifuged for 10 minutes at 4000rpm. The organic layer was decanted and used for HPLC injection. Sampleswere analyzed on a Chiralpak IC 4.6-mm ID×25-cm column with aHeptane/iPrOH/TFA (98/2/0.1) mobile phase at a flow rate of 1.0 mL/min.

TABLE 1 Enzymatic panel chiral HPLC peak area responses of the selectivehydrolysis and desymmetrization of (±)-diethyl diester 13 to2-(difluoromethyl)-1-(ethoxycarbonyl) cyclopropanecarboxylic acids (14).8.2 min 8.5 min 9.3 min 9.8 min 10.8 min 12.8 min Diester DiesterMonoacid Monoacid Monoacid Monoacid Sample # Enzyme (S)-13 (R)-13(1R,2S)-14 (1S,2R)-14 (1S,2S)-14 (1R,2R)-14 1 Lipase - Rhizomucor miehei73.0 N/D 22.7 <4.4 N/D N/D 2 Esterase- Bacillus stearothermophilus 69.75.1 6.0 10.0 4.8 4.4 3 Esterase- Rabbit liver 24.6 2.1 36.4 25.4 N/D11.5  4 Esterase- Porcine liver 33.7 3.1 21.8 26.8 8.5 6.1 5 PLE Isozyme1 51.0 4.9 20.1 13.0 2.7 8.3 6 PLE Isozyme 2 41.3 3.5 25.5 17.6 2.8 9.47 PLE Isozyme 3 22.5 1.9 12.2 33.9 24.5 4.9 8 PLE Isozyme 4 23.8 2.017.5 34.1 18.1 4.5 9 PLE Isozyme 5 23.7 2.4 18.1 38.5 17.3 N/D 10 PLEIsozyme 6 17.3 1.5 17.6 42.7 19.8 1.2 11 Esterase- Bacillus subtilis18.2 1.6 1.8 37.7 35.9 4.6 12 Esterase- Bacillus stearothermophilus 36.33.3 9.8 47.6 3.0 N/D 13 Esterase- Paeriibacillus barcinonensis 28.6 2.6N/D 32.7 36.2 N/D 14 Esterase- Pyrobaculum calidifontis 41.7 3.6 11.97.9 4.2 30.7  Chemical N/D N/D 36.1 35.9 7.2 7.7 Hydrolysis

Example 3b: Preparation of Esterase, “yvaK” Enzyme

An esterase ‘yvaK’ enzyme from Bacillus subtilis was envisioned forselective one step reactive resolution and desymmetrization of(±)-diethyl diester (13) or desymmetrization of (R)-propyl diester (7)due to its high selectivity. The following procedure describes thecreation and preparation of ‘yvaK’ esterase for use as cell-free lysate.

Construction of yvaK Expression Plasmid and Cell Stocks

The following nucleotide sequence was synthesized in pUC57 for use inthe subsequent expression of the desired yvaK esterase. Restrictionendonuclease sequences of NdeI and BamHI were placed on the respective5′ and 3′ ends of the gene sequence for use in ligation to a pET21aexpression vector. Alternatively, the restriction digested yvaK gene canbe ligated into the pET28a-c expression plasmid using the sameprocedure. Upon successful ligation into the multiple cloning site ofpET21a/pET28a-c at sites NdeI and BamHI, the plasmid was transformed inE. coli strain BL21(DE3) competent cells for expression. During thetransformation, 2 μL of the plasmid prep was added to competent E. colicells and incubated on ice for 30 min, followed by heat shock at 42° C.for 1 min and back on ice for 2 min. 200 μL of SOC media was added tothe transformation mix and incubated at 37° C. for 1 hr. This was platedon pre-warmed LB agar plate containing appropriate antibiotics. Theplate was then incubated overnight at 37° C. to allow colonies to form.These colonies were used to inoculate cultures for the subsequentproduction of cell stocks. Cell stocks were created by mixing equalparts of an overnight (37° C.) culture with pre-sterilized 50:50H₂0:Glycerol and stored at −80° C. until use.

The yvaK esterase gene was restriction digested from the pUC57 vectorand ligated into pEt28a using the above procedure and confirmed bycolony PCR.

Bacillus subtilis BS2, yvaK Esterase DNA with Restriction Sites NdeI andBamHI

Restriction sites: NdeI, BamHI

Name: BS2_Esterase, aka yvaK Gene/insert size: 750 bp

Protein: 246 amino acids, ˜32 kDa Gene ID: BSU33620

Species: Bacillus subtilis subsp. subtilis str. 168

CATatgaaagttgtgacaccaaaaccatttacatttaaaggcggagacaaagcggtgcttttgctgcatggctttacaggaaatacagcggatgtaaggatgctgggacgatatttgaatgaacgcggctatacgtgccacgcgcctcaatatgaaggacatggcgtcccgcctgaagaacttgtacatacggggcccgaagactggtggaaaaacgtaatggatggctatgaatatttaaaatctgaaggttatgagagcattgctgcctgcggactgtcgcttggeggggttttttcgctgaaattgggttacactgtacccataaagggaattgtcccaatgtgcgcaccgatgcatattaagagtgaagaggtcatgtatcaaggcgttctttcatacgctcgcaattacaaaaagtttgaggggaaaagcccggagcaaattgaagaggaaatgaaagaattcgaaaaaacgccgatgaataccctcaaggcgctgcaagacttaattgctgatgtgcggaataatgtcgatatgatttattcaccgacatttgtggtgcaggcccgtcatgaccacatgattaataccgaaagcgccaatattatttacaacgaagtggaaactgatgataaacagctgaaatggtacgaggaatcagggcatgtcattacactcgacaaagaacgtgacctcgtccatcaggatgtgtatgaatttttagagaagctc gattggtaaGGATCC.

Lab-Scale Expression Protocol:

Step 1:

Seed inoculum (10 mL) was grow from the yvaK pET21a BL21(DE3) cellstocks overnight in sterilized Luria Broth (LB) media containing 50μg/mL ampicillin at 37° C. temperature.

Step 2:

Prepare a sterilized, baffled 2 L flask containing 500 mL of LB mediawith 50 μg/mL ampicillin. Inoculate the flask at 1:200 ratio ofovernight seed culture to fermentation broth volume (2.5 mL seed cultureper 500 mL fermentation broth)

Step 3:

Incubate the fermentation flask at 37° C. and 225 rpm. During thisfermentation, periodically monitor the optical density at 600 nm (OD₆₀₀)to determine when the culture has reached the proper growth forinduction. It should require approximately 4 hours of incubation toreach the desired OD₆₀₀ of 0.5-0.8 AU. Upon reaching the desired OD₆₀₀,induce the culture by addition of IPTG to a final culture concentrationof 0.1 mM.

Step 4:

Following induction, adjust the incubation temperature down to 30° C.and 225 rpm, and incubate the culture for 16 hours.

Step 5:

Harvest cells by centrifugation for 30 minutes at 3750 rpm and 4° C.Decant and use the resulting cell pellet in preparation of cell-freelysate.

Cell-Free Lysate Preparation:

Step 1:

Resuspend cell pellet by vortexing in 0.5 M K₂HPO₄ buffer pH 7.0 at aratio of 1:10 resuspension buffer to original culture volume (50 mL ofbuffer for a 500 mL-culture pellet).

Step 2:

Sonicate the resulting slurry on ice, 3 times for 30 seconds allowing 1minute intervals in between to cool the culture.

Step 3:

Centrifuge lysed cell slurry at 7500 rpm, 4° C. for 20 minutes to removeinsoluble cell debris. Decant soluble fraction for use a cell-freelysate.

Fermentation of Esterase Enzyme, yvaK:

A 25 mL starter culture of the esterase enzyme, yvaK, was grown up in LBbroth on a shaker at 37° C., 180 RPM in the presence of kanamycin (50 μgmL⁻¹) for 7 hrs. Fermentation media was prepared in the 2 L fermenter(working volume 1.5 L) by adding 100 mL of 10× M9 salts (below), 1 mL ofautoclaved trace elements (below, autoclave separately), 1 mL ofautoclaved magnesium sulfate (1 M, filter sterilized), 1 mL ofautoclaved antifoam (propylene glycol) and 1 mL of autoclaved yeastextract (1 g in 10 mL). These components were then added to 30 g ofglucose in 100 mL of H₂O, which was autoclaved separately.

10× M9 Salts:

Material Wt Sodium phosphate monobasic, >99% 40 g Potassium phosphatedibasic, ACS reagent, 98% 146 g  Ammonium chloride  5 g Ammoniumsulfate >99% 25 g Citric acid, 99% 10 g Sodium sulfate 20 g 1 L of wateris added to the above ingredients and the mixture is heat sterilized insitu in the fermenter by autoclaving at 121° C. for 30 minutes

Trace Metals:

Material Wt CaCl₂•6 H₂O 0.74 g ZnSO₄•7 H₂O 0.18 g MnSO₄•H₂O, 99%  0.1 gCitric acid, 99% 20.1 g FeCl₃•6 H₂O 16.7 g CuSO₄•5 H₂O  0.1 g CoCl₂•6H₂O, 98% ACS reagent 0.104 g  1 L of water is added to the aboveingredients and the mixture is heat sterilized in situ in the fermenterby autoclaving at 121° C. for 30 minutes

The 25 mL starter culture of yvaK was then added to the fermenter. Thefermenter was allowed to run at 37° C., with continuous stirring (1200RPM) with a constant supply of filter-sterilized air from an air pump(initial rate 2 vessel volumes min⁻¹). The pH was maintained at pH 7 byaddition of 35% v/v solutions of ammonium hydroxide and phosphoric acid,as necessary. After 36 hours, the measured OD₆₀₀ at 36 hours was 17. Theglycerol feed was initiated to provide extra nutrients to the growingcells. The components of the glycerol fed are listed below.

Glycerol Feed:

Material Amount (mL) Glycerol, 99% 60 mL 10 × M9 salts 60 mL Magnesiumsulfate  1 mL of 1M solution Trace Metals  1 mL (Autoclaved separately)Yeast extract, 100 g L⁻¹  1 mL 380 mL of water is added to the aboveingredients and the mixture is heat sterilized in situ in the fermenterby autoclaving at 121° C. for 30 minutes. The glycerol was autoclavedseparately.

After 44 hours and a measured OD₆₀₀ of 23, the temperature of thefermentation was reduced to 25° C. for protein overexpression. At 45hours and a measured OD₆₀₀ of 27, the temperature had stabilized at 25°C. Overexpression of the recombinant yvaK enzyme was started by additionof filter sterilized Isopropyl-β-D-1-thiogalactopyranoside (IPTG) to afinal concentration of 1 mM. The cells were allowed to continue growingunder these conditions overnight for a total fermentation time of 56hours. At the end of the fermentation, indicated by a rise in dissolvedoxygen (DO), the wet cell pellet was collected by centrifugation at 8000RPM for 10 minutes. A total yield of 51.2 g L⁻¹ of wet cell pellet wasobtained from the fermentation.

Example 3c: One-Step Enzymatic Reactive Resolution of (±)-diethyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (13)

In this reaction, yvaK enzyme was prepared as described to yieldcell-free lysate of the enzyme. Into an 500 mL Erlenmeyer flask, 210 mLof sodium phosphate buffer was added (0.5 M, pH 6.9), and combined with15 mL of yvaK cell-free lysate solution. The racemic (±)-diethyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (13, 750 mg) was added tothe flask and set at 30° C. and 125 RPM in a shaker incubator. Thereaction was allowed to proceed for 144 hours, and reaction progress wasmonitored by chiral HPLC. The remaining unreacted diester was removed bytwice extracting into 60 mL of methyl tert-butyl ether (MTBE). Theremaining aqueous phase was pH adjusted to 3 by the addition of 17 mL of5 N hydrochloric acid. The monoacid reaction product was recovered bysubsequent extraction using three 60 mL fractions of MTBE. The organicfractions were combined and filtered through Celite.

The reaction resulted in 97% conversion of the desired (R)-diester (13)to monoacid isomers (1R,2S)-14:(1S,2R)-14:(1S,2S)-14:(1R,2R)-14 withratio of with a ratio of isomers 1:2.7:1.25:0 as determined by chiralHPLC, respectively (HPLC elution order: Isomer 1, (1R,2S)-14; Isomer 2(1S,2R)-14; Isomer 3 (1S,2S)-14; Isomer 4 (1R,2R)-14). From thisenzymatic reaction, 81.8% of all monoacid isomers (14) were isolated asa mixture by extraction with MTBE solvent and concentration on a rotaryevaporator.

Example 4: Two Step Enzymatic Reactive Resolution and Desymmetrizationof (±)-diethyl 2-(difluoromethyl)cyclopropane-1,1-carboxylate (13)

Example 4a: Enzymatic Reactive Resolution of (±)-diethyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (13)

To a solution of RML (lipase from Rhizomucor miehei) (50 mL/g, 27.5 mL)in 0.5 M NaH₂PO₄ buffer (3.3 g/L total aq. volume, 125 mL) was added asolution of (±)-diethyl 2-(difluoromethyl)cyclopropane-1,1-carboxylate(13, 2.328 mmol, 0.55 g) in DMF (10% v/v, 15 mL). The resulting solutionwas shaken in an Erlenmeyer flask at 150 rpm at 30° C. for 95 h. Thereaction was monitored by chiral HPLC [Regis (S,S)-Whelk O1 5/100Kromasil 4.6×25 cm, Heptane/i-PrOH/TFA (98/2/0.1], Detector: UV 230 nm]for consumption of (S)-enantiomer of diester (13). The reaction wasquenched with brine (50 mL) and saturated NaHCO₃ solution and the pH wasadjusted to 9. The aqueous reaction solution was extracted with MTBE(3×150 mL); washed combined organic layers with saturated NaHCO₃solution (1×100 mL) and brine (2×100 mL). The undesired(1R,2S)-2-(difluoromethyl)-1-(ethoxycarbonyl) cyclopropanecarboxylicacid (14) remained in aqueous phase and removed. The combined organiclayers were dried over excess MgSO₄, filtered, and concentrated on arotary evaporator under vacuum to give the resolved (R)-diethyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (13) as a pale yellow oil(0.168 g, 97.4% e.e., 64.4% yield).

Example 4b: Enzymatic Desymmetrization of (R)-diethyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (13)

To a solution of yvaK in 0.5 M NaH₂PO₄ buffer (5:1 culture volresuspension, 45 mL) was added (R)-diethyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (13, 0.635 mmol, 0.15 g).The resulting solution was shaken in an Erlenmeyer flask at 150 rpm at30° C. for 68 h. The reaction was monitored by chiral HPLC (Regis(S,S)-Whelk O1 5/100 Kromasil 4.6×25 cm, Heptane/iPrOH/TFA [98/2/0.1],UV 230 nm) for consumption of (R)-enantiomer of diester (13). Thereaction was quenched with 5N HCl (pH adjust to 2). The aqueous reactionsolution was extracted with MTBE (3×60 mL), with centrifugationfollowing each extraction (10 min at 2500 rpm). The combined organiclayers were washed with brine (2×50 mL), then dried over excess MgSO₄,filtered, and concentrated in vacuo to give(1S,2R)-2-(difluoromethyl)-1-(ethoxycarbonyl)cyclopropanecarboxylic acid(14) as an amber oil (0.109 g, 99.4% e.e., 84.2% yield).

Comparative Example 4a: Separation of (±)-diethyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (13) enantiomers

(±)-Diethyl 2-(difluoromethyl)cyclopropane-1,1-carboxylate (13) wasdissolved in Heptane/i-PrOH/TFA (98:2:0.1) and made a stock solutionwith concentration of 40 mg/mL for purification/separation of bothenantiomers of 13 by preparative HPLC system (Column: ChiralPak IC, 5μ21×250 mm; Mobile Phase: Heptane/i-PrOH/TFA (98/2/0.1), Solvent system:Isocratic; Detector: UV 215 nm, Column temperature: Ambient, 19-23° C.;Injection Volume: 2 mL, Run time: 20 min). Both (R) and (S)-enantiomersof 13 were collected in two fractions and the combined fractionsconcentrated on a rotary evaporator under vacuum to afford 0.94 g ofIsomer 1, (S)-diethyl 2-(difluoromethyl)cyclopropane-1,1-carboxylate((S)-13, Isomer 1) in >99.5% e.e., and 0.89 g of Isomer 2, (R)-diethyl2-(difluoromethyl)cyclopropane-1,1-carboxylate ((R)-13, Isomer 2)in >99.5% e.e.

Example 5: Enzymatic Reactive Resolution Screening of (±)-dimethyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (15), (±)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (7) and (±)-dibutyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (17)

A range of esters were synthesized as alternatives to the ethyl diester(13) (Example 4) to ascertain if increases in selectivity could beachieved. Racemic methyl (15), propyl (7) and butyl (17) diesters weresynthesized for evaluation in the hydrolysis reaction of racemicdiester. Several enzymes; AH-45, AH-46, and DSM PLE 444 showedimprovements in selectivity during preliminary screening were evaluatedfor their applicability.

Example 5a: Methyl Diester (15)

Screening of methyl diester (15) yielded a number of potential enzymeswhich were screened for their selectivity in diester hydrolysis. Theresults of this optimization are detailed in the following section.

DSM PLE 444 Mediated Desymmetrization of Methyl Diester (15)

DSM PLE 444 was identified as a potential enzyme to deliver the required(1S,2R)-monoacid product directly. Reactions were carried out over arange of pH's to determine if the E-value could be improved sufficientlyto make this a viable option. The reaction at pH 6 gave the highestselectivity toward the desired monoacid by the following procedure.

To a 3-neck 50 mL round bottomed flask, KH₂PO₄ buffer (0.1 M, 20 mL) wascharged. Enzyme DSM PLE 444 was charged followed by addition ofsubstrate (100 mg). The reactions were stirred at 1000 rpm with magneticfleas. The reactions were allowed to run overnight and worked up foranalysis. The pH was adjusted to 1.85 by addition of 2 M HCl. Theaqueous was extracted with MTBE (2×10 mL). The combined organics wereconcentrated in vacuo, taken up in 95/5 heptane/EtOH, filtered throughMgSO₄ and concentrated.

AH-45 Mediated Resolution of Methyl Diester (15)

To a COC vial, 0.1 M KH₂PO₄ buffer (pH 6, 10 mL) was charged. EnzymeAH-45 (50 μL) was charged followed by addition of dimethyl estersubstrate (15, 50 mg). The reactions were shaken at 200 RPM for 20hours, and then worked up by adjusting the pH to 1.85 by addition of 2 MHCl. The aqueous was extracted with MTBE (2×10 mL). The emulsion wasfiltered through Celite, which was washed with MTBE. The combinedorganics were concentrated in vacuo, taken up in 95/5 hep/EtOH, filteredthrough MgSO₄ and concentrated.

Example 5b: Propyl Diester (7)

Screening of propyl diester (7) yielded a number of potential enzymeswhich were screened for their selectivity in diester hydrolysis. Theresults of this optimization are detailed in the following section.

AH-45 Mediated Resolution of Propyl Diester (7)

To a COC vial, 0.1 M KH₂PO₄ buffer (pH 7, 10 mL) was charged. EnzymeAH-45 (50 μL) was charged followed by addition of dipropyl estersubstrate (7, 50 mg). The reactions were shaken at 200 RPM for 20 hours,and then worked up by adjusting the pH to 1.85 by addition of 2 M HCl.The aqueous was extracted with MTBE (2×10 mL). The emulsion was filteredthrough Celite, which was washed with MTBE. The combined organics wereconcentrated in vacuo, taken up in 95/5 hep/EtOH, filtered through MgSO₄and concentrated. This reaction was carried out in the Ca(OAc)₂ bufferand the rate of reaction was improved significantly.

AH-46 Mediated Resolution of Propyl Diester (7)

To a COC vial, 0.1 M KH₂PO₄ buffer (pH 7, 10 mL) was charged. EnzymeAH-46 (50 μL) was charged followed by addition of dipropyl estersubstrate (7, 50 mg). The reactions were shaken at 200 RPM for 20 hours,and then worked up by adjusting the pH to 1.85 by addition of 2 M HCl.The aqueous was extracted with MTBE (2×10 mL). The emulsion was filteredthrough Celite, which was washed with MTBE. The combined organics wereconcentrated in vacuo, taken up in 95/5 hep/EtOH, filtered through MgSO₄and analyzed by HPLC.

Example 5c: Butyl Diester (17)

Screening of butyl diester (17) yielded a number of potential enzymeswhich were screened for their selectivity in diester hydrolysis. Theresults of this optimization are detailed in the following section.

AH-45 Mediated Resolution of Butyl Diester (17)

To a COC vial, 0.1 M KH₂PO₄ buffer (pH 7, 10 mL) was charged. EnzymeAH-45 (50 μL) was charged followed by addition of dibutyl estersubstrate (17, 50 mg). The reactions were shaken at 200 RPM for 20hours, and then worked up by adjusting the pH to 1.85 by addition of 2 MHCl. The aqueous was extracted with MTBE (2×10 mL). The emulsion wasfiltered through Celite, which was washed with MTBE. The combinedorganics were concentrated in vacuo, taken up in 95/5 hep/EtOH, filteredthrough MgSO₄ and concentrated

AH-46 Mediated Resolution of Butyl Diester (17)

To a COC vial, 0.1 M KH₂PO₄ buffer (pH 8, 10 mL) was charged. EnzymeAH-46 (50 μL) was charged followed by addition of dibutyl estersubstrate (17, 50 mg). The reactions were shaken at 200 RPM for 20hours, and then worked up by adjusting the pH to 1.85 by addition of 2 MHCl. The aqueous was extracted with MTBE (2×10 mL). The emulsion wasfiltered through Celite, which was washed with MTBE. The combinedorganics were concentrated in vacuo, taken up in 95/5 hep/EtOH, filteredthrough MgSO₄ and concentrated.

Example 6: Enzymatic Reactive Resolution and Desymmetrization to Form(1S,2R)-2-(difluoromethyl)-1-(propoxycarbonyl)cyclopropanecarboxylicacid dicyclohexylamine salt (8)

Example 6a: Enzymatic Reactive Resolution of (±)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (7) Via AH-45

0.1 M sodium citrate buffer, pH 5.75 (800 mL, 40 vol) was charged to a3-neck 2-L RBF equipped with a calibrated pH probe, pH stat additionline, baffle and a mechanical stirrer. The reaction mixture was warmedto 20° C. before AH-45 (20 g, 100 wt % wrt substrate) was added. Crude(±)-dipropyl 2-(difluoromethyl)cyclopropane-1,1-carboxylate (7) (20 g)was subsequently added and the reaction was stirred at 400 rpm.

Samples (2 mL) were removed, adjusted to acidic pH by addition of 10drops of 2 M HCl, then extracted with MTBE (2 mL). The organic layer wasconcentrated in vacuo, then the residue was taken up in 95/5 (v/v)Hex/EtOH (1 mL) which was dried over Na₂SO₄ and analyzed on HPLC.

After 72 h the reaction had reached 94% diester ee and was worked up.The reaction pH was adjusted to pH 8.4 by addition of 15% (w/w) Na₂CO₃solution. The reaction mixture was extracted with MTBE (2×100 mL). Theemulsified organic layers were transferred to falcon tubes andcentrifuged at 10000 g for 10 mins. The MTBE layers were decanted off,combined, washed with sat. bicarb (40 mL) and water (40 mL). The organiclayer was then concentrated in vacuo to give 7 as a yellow oil (7.58 g,94.8% e.e., 96% wt/wt by 1H NMR assay). Expected yield=9.4 g,Recovered=7.27 g (corrected for assay), % yield=77.

Example 6b(i): Desymmetrization of (R)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate ((R)-7) Via yvaK

0.1 M sodium phosphate buffer, pH 8.5 (290 mL) was charged to a 3-neck1-L RBF equipped with a calibrated pH probe, pH stat addition line,baffle and a mechanical stirrer. The reaction mixture was warmed to 20°C. before yvaK freeze dried whole cells (6.3 g, 86 wt % wrt substrate)was added. The pH was then re-adjusted to 8.5. (R)-cyclopropyl diester 7(7.58 g, 94.8% e.e., 96% purity by 1H NMR assay) in MTBE (7.6 mL) wassubsequently added and the reaction was stirred at 400 rpm.

Samples were taken periodically to check reaction progress. The analysiswas performed using the achiral method. A sample of the reaction mixture(0.5 mL) was diluted with EtOH (0.5 mL) and THF (0.25 mL). The samplewas then centrifuged at 13200 rpm for 2 mins. The supernatant wasremoved and filtered through a 0.2 Lm filter. The sample was thenanalyzed directly on HPLC.

After 21 h reaction was deemed complete. The pH was adjusted to 1.5 byaddition of 20% HCl solution. Toluene (40 mL) was added followed byCelite (6.3 g) and the reaction mixture stirred for 30 mins. Thesuspension was then filtered on a sintered funnel (filtration slow,Celite resembled chewing gum). The filtrate was transferred toseparating funnel, and the toluene layer was separated off. The Celitewas rinsed with toluene (40 mL) which was then used for 2nd extractionof the aqueous layer. The Celite cake was then transferred to a flaskand slurried overnight with MTBE (100 mL). Concentration of MTBE layeryielded a red oil (2.4 g).

Following on from the DCHA salt screening reactions it was deemed thatsalt formation would be carried out in MTBE/Heptane assolvent/antisolvent. The toluene layer was combined with the residuefrom the MTBE layer and concentrated in vacuo. The residue was azeodriedfrom toluene (2×150 mL), then the residue was taken up in MTBE (50 mL),filtered to remove solid particles and concentrated to afford (1S,2R)-7as a reddish oil (4.04 g, 96.8% e.e., 89% purity by ¹H NMR assay).

Example 6b(ii): Desymmetrization of (R)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate ((R)-7)

In the following procedure, a one-step resolution of enriched(R)-dipropyl ester (7) was performed to yield (1S,2R)-monoacid as themajor product species. In this reaction, a 500 mL culture pellet of yvaKenzyme was resuspended in 25 mL of KH₂PO₄ buffer (0.5 M pH 7.0). Theresuspension was sonicated 3 times in 30 second intervals over ice. Thelysate solution was then centrifuged at 7500 RPM, 4° C. for 15 minutes,and the supernate was retained as the cell-free lysate solution. To thissolution of yvaK in 0.5M KH₂PO₄ buffer (0.035 g [71% wt/wt], 13.4 mL)was added a solution of enriched (R)-dipropyl ester (9, 0.189 mmol,0.050 g) in MTBE (3.33 mL/g, 0.17 mL). The resulting opaque solutionstirred vigorously at room temperature (22° C.) for 98 h. Reactionmixture was quenched with 5N HCl (pH adjust to <2), then extracted withMTBE (2×10 mL), with centrifugation following each extraction (10 min at2500 RPM). The combined organic layers were dried over excess MgSO₄,filtered, and concentrated in vacuo (35° C. water bath) to give(1S,2R)-monoacid ((1S,2R)-6, 0.0422 g) as an amber oil [crude](98.8%e.e., 97% wt/wt by ¹H-NMR, 97% recovery).

Example 6c: Formation of(1S,2R)-2-(difluoromethyl)-1-(propoxycarbonyl)-cyclopropanecarboxylicacid dicyclohexylamine salt (8)

Monoacid (1S,2R)-6 (4.04 g, 96.8% e.e., 89% assay by NMR), prepared asdescribed in Example 6b(i), was dissolved in MTBE (3.6 mL, 1 vol) withstirring. DCHA (3.00 g, 1 eq) was added in slowly to the solution. Asheptane (22 mL) was about to be added, the DCHA salt spontaneouslycrystallised from solution to give a solid mass. Heptane was added andthe solid broken up and stirred for 1 h. The solid was filtered; howeverthe solid contained fine white solid and brown coloured lumps. The solidwas transferred to a flask and slurried in MTBE (10 mL) for 1 h, thenfiltered to leave a white solid which was dried on the filter (3.3 g).Yield too low. The solids and filtrates were combined and concentrated,then the solid was taken up in MTBE (3.6 mL) and heated to 40° C. togive a mobile slurry. Heptane (22 mL) warmed to 40° C. was then added tothe suspension and was stirred for 2 h. The suspension was allowed tocool to room temperature overnight. The suspension was then filtered anddried on the filter to afford (1S,2R)-8 as a white solid (4.0 g, 98.0%e.e.).

Example 6d: Enzymatic Reactive Resolution of (±)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate (7) Via AH-45

0.1 M calcium acetate buffer, pH 7.5 (265 mL, 10.6 vol) was charged to a500-mL RBF equipped with a calibrated pH probe, pH stat addition line,temperature probe and mechanical stirrer. To the buffer solution werecharged AH-45 (12.5 g, 50 wt %) and crude (±)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate ((±)-7, 25 g). Thereaction was stirred at 300 rpm, controlled at 19-24° C., and monitoredby chiral HPLC [Phenomenex Lux 3 [m Cellulose-2, 250×4.6 mm,Heptane/i-PrOH/TFA (98.5/1.5/0.1], Detector: UV 210 nm].

After 90 h, the reaction had reached 99.8% diester e.e. and was adjustedto pH 1.5 by addition of 20% (w/w) HCl solution. Celite (6.7 g, 25 wt %)was added to the reaction mixture and stirred, followed by MTBE (50 mL,2 vol) and stirred. The biphasic mixture was filtered and the filtercake was rinsed with MTBE (3×50 mL). The resulting organic filtrate wasused to extract the aqueous. The organic phases were combined and washedwith 5% (w/w) sodium bicarbonate solution (3×50 mL), dried over MgSO₄,filtered, and concentrated in vacuo to give (R)-7 as a dark yellow oil(9.50 g, 99.6% e.e., 93.3 wt % by qNMR assay). Expected yield=10.95 g,Recovered=8.86 g (corrected for assay), % yield=82.

Example 6e: Desymmetrization of (R)-dipropyl2-(difluoromethyl)cyclopropane-1,1-carboxylate ((R)-7) Via BsteE

0.1 M sodium phosphate buffer, pH 8.3 (117 mL, 14 vol) was charged to a500 mL RBF equipped with a pH probe, pH stat addition line, temperatureprobe and mechanical stirrer. The solution was controlled at 45-50° C.before charging BsteE pretreated cell lysate solution (30 mL, 3.57 vol)followed by (R)-cyclopropyl diester (R)-7 (8.4 g crude, 7.8 g assayadjusted, 99.6% e.e.), prepared as described in example 6d. The reactionwas stirred at 400 rpm, controlled at 45-50° C., and monitored by chiralHPLC.

After 24 h, the reaction had reached >99 PA % monoacid (wrt diester) andwas cooled to 19-24° C., pH was adjusted to 1.5 by addition of 20% (w/w)HCl solution, and diluted with MTBE (50 mL, 6 vol). The biphasic mixturewas filtered and the filter cake was rinsed with MTBE (2×50 mL). Theresulting organic filtrate was used to extract the aqueous. The organicphases were combined and washed with purified water (50 mL), dried overMgSO₄, filtered, and concentrated in vacuo to give (1S,2R)-6 as a darkyellow oil (6.79 g, 99.3% e.e., 93.7 wt % by qNMR assay). Expectedyield=6.59 g, Recovered=6.36 g (corrected for assay), % yield=97.

Example 6f: Formation of(1S,2R)-2-(difluoromethyl)-1-(propoxycarbonyl)-cyclopropanecarboxylicacid dicyclohexylamine salt (8)

Monoacid (1S,2R)-6 (1.09 g crude, 1.02 g assay adjusted, 99.3% e.e.),prepared as described in Example 6e, was charged to a 25 mL flaskequipped with stir bar, followed by MTBE (1.1 mL, 1 vol) and n-heptane(6.6 mL, 6 vol). The mixture was heated to 40-45° C. and mixed until(1S,2R)-6 dissolved completely. Dicyclohexylamine (0.96 mL, 1 eq) wasadded slowly dropwise to the reaction solution. The reaction slurry washeld at temperature and mixed for 1 h, then cooled to ambienttemperature (19-24° C.) and mixed for 16 h. The reaction slurry wassubsequently cooled to 5-10° C. and held at temperature for 6 h, thenfiltered. The filter cake was washed with a 6:1 n-heptane:MTBE solution(2×1 mL), then dried in a vacuum oven (35-40° C., 35-65 torr) for 16 h,to afford (1S,2R)-8 as a white solid (1.42 g, 99.9% e.e., 98.2 wt % byqNMR assay). Expected yield=1.77 g, Recovered=1.39 g (corrected forassay), % yield=79.

Example 7: Curtius Rearrangement to form (1R,2R)-propyl1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropanecarboxylate (12)

Example 7a: Salt Break

A 100-mL round bottom flask, equipped with a Teflon coated magnetic stirbar, was charged with the above dicyclohexylamine salt (1S,2R)-8 (4.0 g,9.91 mmol) and MTBE (30 mL). To this suspension was added a 15% H₃PO₄solution (w/w, 18 mL) and the resulting mixture was stirred at roomtemperature for 15 min. The resulting solution was poured into a 125-mLseparatory funnel and the layers were cut. The top organic layer waswashed with an additional 2.5 mL 15% H₃PO₄ and the layers cut. Theorganic layer was then washed with sat. aq. NaCl, the layers cut, andthe organics dried over MgSO₄. After filtration of the MgSO₄, thesolvent was removed in vacuo to give 2.54 g free acid (1S,2R)-6 as aclear oil.

Example 7b: Curtius Rearrangement

A separate 100-mL three-necked flask equipped with a mechanical stirrerand pressure equalizing addition funnel, was charged with t-BuOH (16.6mL), free acid (1S,2R)-6 (2.54 g, 11.43 mmol), triethylamine (5.26 g,7.25 mL, 52.02 mmol). The mixture was then heated to 80-84° C. (bathtemperature). DPPA (2.43 g, 1.9 mL, 8.82 mmol) was added slowly using asyringe pump, and the mixture was allowed to stir for an additional NLT6 hours after complete addition. The solvent removed under reducedpressure and the crude oil was dissolved in 60 mL MTBE and added to aseparatory funnel. The organic phase was first washed with 5% citricacid (2×30 mL) and the layers cut. The organic phase was then washedwith sat. aq. NaHCO₃ (2×30 mL) and the layers cut. The organics werethen washed with H₂O (2×30 mL) and the layers cut. The organic phase wasdried over Na₂SO₄, filtered, and concentrated to provide 2.45 g of(1R,2R)-propyl1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropanecarboxylate(12) as a yellow-orange oil, which was used as is in next step.

Example 8: Hydrolysis to form(1R,2R)-1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropanecarboxylic acid (1)

A solution of (1R,2R)-propyl1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropanecarboxylate(12) (2.43 g, 8.28 mmol), prepared as described above, in acetonitrile(13 mL) was cooled to 5° C., then treated with a solution of LiOH (0.60g, 24.85 mmol, 3.0 equiv) in water (13 mL), added over 7 minutes. Themixture was stirred at ambient temperature overnight. Upon reactioncompletion, 15% aqueous citric acid was added to achieve a pH of 4-4.5.The mixture was concentrated under vacuum to remove the acetonitrile,and solid NaCl was added to make a saturated solution. The resultingslurry was mixed overnight at ambient temperature, filtered and washedwith 2 mL water to afford 7.5 g of wet cake, which was crystallized fromwater and dried in a vacuum oven to afford 1.1 g of(1R,2R)-1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropanecarboxylicacid (1).

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. The contents of allreferences, patents, and published patent applications, and patentapplications cited throughout this application are incorporated hereinby reference.

1-57. (canceled)
 58. A method according to the following reactionscheme:

wherein R is alkyl; t-BuOH is tert-butyl alcohol; Boc istert-butoxycarbonyl; and DPPA is diphenylphosphoryl azide.
 59. Themethod of claim 58, wherein R is ethyl, propyl, or butyl.
 60. The methodof claim 59, wherein R is n-propyl.
 61. The method of claim 58, whereinthe starting material has the following structure:


62. The method of claim 58, wherein the hydrolysis step comprisestreatment with aqueous LiOH.
 63. A compound having the followingstructure, or a pharmaceutically acceptable salt thereof:


64. The compound of claim 63, wherein the compound has the followingstructure:


65. The compound of claim 64, wherein R is ethyl, propyl, or butyl. 66.The compound of claim 65, wherein R is n-propyl.
 67. The compound ofclaim 64, wherein the compound has the following structure:


68. The compound of claim 64, wherein the compound has the followingstructure:


69. The compound of claim 68, wherein R is ethyl, propyl, or butyl. 70.The compound of claim 69, wherein R is n-propyl.
 71. A compound havingthe following structure, or a pharmaceutically acceptable salt thereof:

wherein R is alkyl; and Boc is tert-butoxycarbonyl.
 72. The compound ofclaim 71, wherein R is ethyl, propyl, or butyl.
 73. The compound ofclaim 72, wherein R is n-propyl.
 74. The compound of claim 71, whereinthe compound has the following structure:


75. The compound of claim 74, wherein R is ethyl, propyl, or butyl. 76.The compound of claim 75, wherein R is n-propyl.
 77. A compound havingthe following structure, or a pharmaceutically acceptable salt thereof:

wherein Boc is tert-butoxycarbonyl.
 78. The compound of claim 77,wherein the compound has the following structure: